title: Temporal profiling of the adipocyte proteome during differentiation using a five-plex SILAC based strategy.
abstract: The adipose tissue has important secretory and endocrine functions in humans. The regulation of adipocyte differentiation has been actively pursued using transcriptomic methods over the last several years. Quantitative proteomics has emerged as a promising approach to obtain temporal profiles of biological processes such as differentiation. Stable isotope labeling with amino acids in cell culture (SILAC) is a simple and robust method for labeling proteins in vivo. Here, we describe the development and application of a five-plex SILAC experiment using four different heavy stable isotopic forms of arginine to study the nuclear proteome and the secretome during the course of adipocyte differentiation. Tandem mass spectrometry analysis using a quadrupole time-of-flight instrument resulted in identification of a total 882 proteins from these two proteomes. Of these proteins, 427 were identified on the basis of one or more arginine-containing peptides that allowed quantitation. In addition to previously reported molecules that are differentially expressed during the process of adipogenesis (e.g., adiponectin and lipoprotein lipase), we identified several proteins whose differential expression during adipocyte differentiation has not been documented previously. For example, THO complex 4, a context-dependent transcriptional activator in the T-cell receptor alpha enhancer complex, showed highest expression at middle stage of adipogenesis, while SNF2 alpha, a chromatin remodeling protein, was downregulated upon initiation of adipogenesis and remained so during subsequent time points. This study using a 5-plex SILAC to investigate dynamics illustrates the power of this approach to identify differentially expressed proteins in a temporal fashion.
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.|Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Henrik
authors.initials: H
authors.lastname: Molina
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.|Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Travis
authors.initials: T
authors.lastname: Ruch
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Jae-Woo
authors.initials: JW
authors.lastname: Kim
authors.affiliation: Center for Experimental BioInformatics (CEBI), Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Mortensen
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Tamara
authors.initials: T
authors.lastname: Otto
authors.affiliation: Institute of Bioinformatics, International Technology Park, Bangalore 560066, India.
authors.forename: Anuradha
authors.initials: A
authors.lastname: Nalli
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Qi-Qun
authors.initials: QQ
authors.lastname: Tang
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: M Daniel
authors.initials: MD
authors.lastname: Lane
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.|Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.|Institute of Bioinformatics, International Technology Park, Bangalore 560066, India.
authors.forename: Raghothama
authors.initials: R
authors.lastname: Chaerkady
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.|Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.|Departments of Pathology and Oncology, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Akhilesh
authors.initials: A
authors.lastname: Pandey
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism*; D050156:Adipogenesis; D000818:Animals; D001391:Azo Compounds / Q000494:pharmacology; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D005786:Gene Expression Regulation; D007554:Isotopes; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D040901:Proteomics / Q000379:methods*; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
chemical_list: D001391:Azo Compounds; D007554:Isotopes; D014357:Trypsin; C011049:oil red O